title: Pan1 is an intrinsically disordered protein with homotypic interactions.
abstract: The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp(312) and Trp(642) are each in an EH domain, Trp(957) is in the central region, and Trp(1280) is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland, 21218.
authors.forename: B D
authors.initials: BD
authors.lastname: Pierce
authors.forename: Dmitri
authors.initials: D
authors.lastname: Toptygin
authors.forename: Beverly
authors.initials: B
authors.lastname: Wendland
mesh_terms: D005454:Fluorescence Polarization; D005656:Fungal Proteins; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D008840:Microfilament Proteins; C111870:PAN1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan
keywords: EH domain; Pan1; intrinsically disordered protein; time-resolved fluorescence anisotropy; tryptophan fluorescence quenching